VEGF antagonist formulations for intravitreal administration

ABSTRACT

Ophthalmic formulations of a vascular endothelial growth factor (VEGF)-specific fusion protein antagonist are provided suitable for intravitreal administration to the eye. The ophthalmic formulations include a stable liquid formulation and a lyophilizable formulation. Preferably, the protein antagonist has an amino acid sequence of SEQ ID NO:4.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/329,770, filed Dec. 19, 2011, which issued asU.S. Pat. No. 8,481,046, which is a continuation application of U.S.patent application Ser. No. 12/833,417, filed Jul. 9, 2010, which issuedas U.S. Pat. No. 8,092,803 on Jan. 10, 2012, which is a continuationapplication of U.S. patent application Ser. No. 12/560,885, filed Sep.16, 2009, which issued as U.S. Pat. No. 7,807,164 on Oct. 5, 2010, whichis a divisional application of U.S. patent application Ser. No.11/818,463, filed 14 Jun. 2007, which issued as U.S. Pat. No. 7,608,261on Oct. 27, 2009, which claims the benefit under 35 U.S.C. §119(e) ofU.S. Provisional Application No. 60/814,484, filed 16 Jun. 2006, whichapplications are each hereby incorporated by reference.

BACKGROUND OF INVENTION

1. Field of the Invention

The present invention is directed to pharmaceutical formulationssuitable for intravitreal administration comprising agents capable ofinhibiting vascular endothelial growth factor (VEGF), and to methods formaking and using such formulations. The invention includes liquidpharmaceutical formulations having increased stability, as well asformulations that may be lyophilize and reconstituted for intravitrealadministration.

2. Statement of Related Art

Vascular endothelial growth factor (VEGF) expression is nearlyubiquitous in human cancer, consistent with its role as a key mediatorof tumor neoangiogenesis. Blockade of VEGF function, by binding to themolecule or its VEGFR-2 receptor, inhibits growth of implanted tumorcells in multiple different xenograft models (see, for example, Gerberet al. (2000) Cancer Res. 60:6253-6258). A soluble VEGF-specific fusionprotein antagonist, termed a “VEGF trap” has been described (Kim et al.(2002) Proc. Natl. Acad. Sci. USA 99:11399-404; Holash et al. (2002)Proc. Natl. Acad. Sci. USA 99:11393-8), which applications arespecifically incorporated by reference in their entirety.

Ophthalmic formulations are known, see for example, U.S. Pat. Nos.7,033,604 and 6,777,429. An ophthalmic formulation of a VEGF antibody isdescribed in U.S. Pat. No. 6,676,941.

Lyophilization (freeze drying under controlled conditions) is commonlyused for long-term storage of proteins. The lyophilized protein issubstantially resistant to degradation, aggregation, oxidation, andother degenerative processes while in the freeze-dried state (see, forexample, U.S. Pat. No. 6,436,897).

BRIEF SUMMARY OF THE INVENTION

Stable formulations of a VEGF-specific fusion protein antagonist areprovided. Pharmaceutically acceptable formulations are provided thatcomprise a VEGF “trap” antagonist with a pharmaceutically acceptablecarrier. In specific embodiments, liquid and lyophilized formulationsare provided.

In a first aspect, a stable liquid ophthalmic formulation of aVEGF-specific fusion protein antagonist is provided, comprising a fusionprotein that comprises a receptor component consisting essentially of animmunoglobulin-like (Ig) domain 2 of a first VEGF receptor and Ig domain3 of a second VEGF receptor, and a multimerizing component (also termeda “VEGF trap”). In a specific embodiment of the VEGF-specific fusionprotein antagonist, the first VEGF receptor is Flt1 and the second VEGFreceptor is Flk1 or Flt4. In a more specific embodiment the fusionprotein has the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.Preferably, the VEGF antagonist is a dimer comprising two fusionproteins of SEQ ID NO:4.

In one aspect, a stable liquid ophthalmic formulation is provided thatcomprises 1-100 mg/ml VEGF-specific fusion protein antagonist, 0.01-5%of one or more organic co-solvent(s), 30-150 mM of one or more tonicityagent(s), 5-40 mM of a buffering agent, and optionally, 1.0-7.5% of astabilizing agent, pH between about 5.8-7.0.

In one or more specific embodiments, the organic co-solvent may bepolysorbate, for example, polysorbate 20 or polysorbate 80, polyethyleneglycol (PEG), for example, PEG 3350, or propylene glycol, or acombination thereof; the tonicity agent may be, for example, sodiumchloride or potassium chloride; the stabilizing agent may be sucrose,sorbitol, glycerol, trehalose, or mannitol; and the buffering agent maybe, for example, phosphate buffer. In a specific embodiment, thephosphate buffer is a sodium phosphate buffer.

In various embodiments, the organic co-solvent is polysorbate and/orPEG, the stabilizing agent is sucrose, the buffering agent is phosphatebuffer, and the tonicity agent is sodium chloride.

More specifically, the stable liquid ophthalmic formulation comprisesabout 40-50 mg/ml of the VEGF antagonist (SEQ ID NO:4), about 10 mMphosphate buffer, 0.01-3% polysorbate and/or PEG, 40-135 mM sodiumchloride, and optionally 5.0% sucrose, pH about 6.2-6.3.

In a specific preferred embodiment, the stable liquid ophthalmicformulation comprises about 50 mg/ml of the VEGF antagonist (SEQ IDNO:4), 10 mM sodium phosphate buffer, 50 mM sodium chloride, 0.1%polysorbate, and 5% sucrose, pH about 6.2-6.3.

In a specific preferred embodiment, the stable liquid ophthalmicformulation comprises about 50 mg/ml of the VEGF antagonist (SEQ IDNO:4), 10 mM sodium phosphate buffer, 50 mM sodium chloride, 3% PEG, and5% sucrose, pH about 6.2-6.3.

In a specific preferred embodiment, the stable liquid ophthalmicformulation comprises about 40 mg/ml of the VEGF antagonist (SEQ IDNO:4), 10 mM sodium phosphate buffer, 40 mM sodium chloride, 0.03%polysorbate, and 5% sucrose, pH about 6.2-6.3.

In a specific preferred embodiment, the stable liquid ophthalmicformulation comprises about 40 mg/ml of the VEGF antagonist (SEQ IDNO:4), 10 mM sodium phosphate buffer, 135 mM sodium chloride, and 0.03%polysorbate, pH about 6.2-6.3.

In another aspect, a stable liquid ophthalmic formulation is providedthat comprises 1-100 mg/ml VEGF-specific fusion protein antagonist;0.01-5% of one or more organic co-solvent(s); 5-40 mM of a bufferingagent; and optionally 30-150 mM of one or more tonicity agent(s) and/or1.0-7.5% of a stabilizing agent; having a pH between about 5.8-7.0.

In various embodiments, the VEGF antagonist (SEQ ID NO:4) is present ata concentration of about 10 to about 80 mg/ml. In various embodiments,the VEGF antagonist (SEQ ID NO:4) is present at a concentration of about10, about 20, about 30, about 40, about 50, about 60, about 70, or about80 mg/ml. In a preferred embodiment, the VEGF antagonist (SEQ ID NO:4)is present at a concentration of about 40 mg/ml.

In another embodiment, the stabilizing agent is selected from one ormore of sucrose, sorbitol, glycerol, trehalose, and mannitol.

In another embodiment, the organic co-solvent is selected from one ormore of polysorbate, for example, polysorbate 20 or polysorbate 80,polyethylene glycol (PEG), for example, PEG 3350, and propylene glycol.

In another embodiment, the buffer is a phosphate buffer, for example,sodium phosphate.

In another embodiment, the tonicity agent is a salt, for example, sodiumchloride.

In one embodiment, the stable liquid ophthalmic formulation comprises 10mM sodium phosphate buffer, about 0.03 to about 0.1% polysorbate and/orabout 3% PEG or propylene glycol, about 40 mM sodium chloride, and about5% sucrose. In a specific embodiment, the stable liquid ophthalmicformulation comprises 10 mM sodium phosphate buffer, about 0.03%polysorbate, about 40 mM sodium chloride, and about 5% sucrose. Inanother specific embodiment, the pH of the formulation is about 6.2 toabout 6.3. In another specific embodiment, the pH is achieved by mixingmono- and dibasic sodium phosphate to the desired pH without acid/basetitration.

In a specific embodiment, the stable liquid ophthalmic formulationconsists essentially of a VEGF antagonist (SEQ ID NO:4) at 40 mg/ml, 10mM sodium phosphate buffer, polysorbate at 0.03%, sodium chloride at 40mM, and sucrose at 5%, pH 6.2-6.3.

In another aspect, a stable liquid ophthalmic formulation is providedthat comprises about 10 to about 80 mg/ml VEGF antagonist, about 10 mMsodium phosphate buffer, about 0.03% polysorbate, and about 135 mMsodium chloride, pH 6.2 to 6.3.

In various embodiments, the VEGF antagonist (SEQ ID NO:4) is present ata concentration of about 10 to about 80 mg/ml. In various embodiments,the VEGF antagonist (SEQ ID NO:4) is present at a concentration of about10, about 20, about 30, about 40, about 50, about 60, about 70, or about80 mg/ml. In a specific embodiment, the VEGF antagonist (SEQ ID NO:4) ispresent at a concentration of about 40 mg/ml.

In one embodiment, the stable liquid ophthalmic formulation comprises 40mg/ml of VEGF antagonist (SEQ ID NO:4), 10 mM sodium phosphate buffer,0.03% polysorbate, and 135 mM sodium chloride at pH 6.2-6.3. In aspecific embodiment, the stable liquid ophthalmic formulation consistsessentially of 40 mg/ml of VEGF antagonist (SEQ ID NO:4), 10 mM sodiumphosphate buffer, 0.03% polysorbate, and 135 mM sodium chloride at pH6.2-6.3.

In another aspect, a lyophilizable formulation of a VEGF antagonist isprovided, wherein upon lyophilization followed by reconstitution, astable liquid ophthalmic formulation as described herein is obtained.

In another aspect, a lyophilizable formulation of a vascular endothelialgrowth factor (VEGF)-specific fusion protein antagonist is provided,comprising 5-50 mg/ml of the VEGF antagonist, 5-25 mM buffer, such asphosphate buffer, 0.01 to 0.15% of one or more of an organic co-solvent,such as polysorbate, propylene glycol and/or PEG, and optionally 1-10%of a stabilizing agent such as sucrose, sorbitol, trehalose, glycerol,or mannitol, pH about 5.8-7.0. In various embodiments, the VEGFantagonist (SEQ ID NO:4) is present at about 5, about 10, about 20,about 30, or about 40 mg/ml. In a specific embodiment, the lyophilizableophthalmic formulation of the invention comprises 20 mg/ml of the VEGFantagonist, 10 mM sodium phosphate buffer, 0.03% polysorbate, 0.1% PEG,and 2.5% sucrose, pH about 6.2-6.3. In further embodiments, thelyophilizable formulation further comprises sodium chloride. In aspecific embodiment, the sodium chloride is present at a concentrationof about 20 mM. In another specific embodiment, the sodium chloride ispresent at a concentration of about 67.5 mM.

In another specific embodiment, the lyophilizable ophthalmic formulationof the invention comprises 20 mg/ml of the VEGF antagonist, 5 mM sodiumphosphate buffer, 0.015% polysorbate, 20 mM sodium chloride, and 2.5%sucrose, pH about 6.2-6.3.

In another embodiment, the lyophilizable ophthalmic formulationcomprises 5 mg/ml, 10 mg/ml, or 40 mg/ml VEGF antagonist, 5 mM sodiumphosphate buffer, 0.015% polysorbate, 20 mM sodium chloride, and 2.5%sucrose, at pH 6.2-6.3. In a specific embodiment, the lyophilizableophthalmic formulation consists essentially of 5 mg/ml, 10 mg/ml, or 40mg/ml VEGF antagonist (SEQ ID NO:4), 5 mM sodium phosphate buffer,0.015% polysorbate, 20 mM sodium chloride, and 2.5% sucrose, at pH6.2-6.3.

In another specific embodiment, the lyophilizable ophthalmic formulationcomprises 20 mg/ml of the VEGF antagonist, 5 mM sodium phosphate buffer,0.015% polysorbate, and 67.5 mM sodium chloride, pH about 6.2-6.3. In amore specific embodiment, the lyophilizable ophthalmic formulationconsists essentially of 20 mg/ml of the VEGF antagonist (SEQ ID NO:4), 5mM sodium phosphate buffer, 0.015% polysorbate, and 67.5 mM sodiumchloride, pH 6.2-6.3.

In another specific embodiment, the lyophilizable ophthalmic formulationcomprises 5 mg/ml, 10 mg/ml, or 40 mg/ml VEGF antagonist, 5 mM sodiumphosphate buffer, 0.015% polysorbate, and 67.5 mM sodium chloride, pHabout 6.2-6.3. In a more specific embodiment, the lyophilizableophthalmic formulation consists essentially of 5 mg/ml, 10 mg/ml, or 40mg/ml VEGF antagonist (SEQ ID NO:4), 5 mM sodium phosphate buffer,0.015% polysorbate, and 67.5 mM sodium chloride, pH about 6.2-6.3.

Generally, the reconstituted formulation is about 2 times theconcentration of the pre-lyophilized formulation, e.g., a 20 mg fusionprotein/ml pre-lyophilized formulation is reconstituted to a finalformulation of 40 mg fusion protein/ml.

Generally, the lyophilized formulation is reconstituted with sterilewater suitable for injection. In one embodiment, the reconstitutionliquid is bacteriostatic water.

In another aspect, the invention features a method of producing alyophilized formulation of a VEGF-specific fusion protein antagonist,comprising subjecting the lyophilizable formulation of the invention tolyophilization to generate a lyophilized formulation. The lyophilizedformulation may be lyophilized by any method known in the art forlyophilizing a liquid.

In another related aspect, the invention features a method of producinga reconstituted lyophilized formulation of a VEGF antagonist, comprisingreconstituting the lyophilized formulation of the invention to areconstituted formulation. In one embodiment, the reconstitutedformulation is twice the concentration of the pre-lyophilizedformulation, e.g., the method of the invention comprises: (a) producinga pre-lyophilized formulation of a VEGF-specific fusion proteinantagonist, (b) subjecting the pre-lyophilized formulation of step (a)to lyophilization; and (c) reconstituting the lyophilized formulation ofstep (b).

The invention further features ophthalmic formulations provided in apre-filled syringe or vial, particularly suitable for intravitrealadministration.

Other objects and advantages will become apparent from a review of theensuing detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is not limited to particular methods, andexperimental conditions described, as such methods and conditions mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting unless indicated, since the scope of the presentinvention will be limited only by the appended claims.

Unless stated otherwise, all technical and scientific terms and phrasesused herein have the same meaning as commonly understood by one ofordinary skill in the art to which the invention belongs. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, thepreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference.

General Description

Safe handling and administration of formulations comprising proteinsrepresent significant challenges to pharmaceutical formulators. Proteinspossess unique chemical and physical properties that present stabilityproblems: a variety of degradation pathways exist for proteins,implicating both chemical and physical instability. Chemical instabilityincludes deamination, aggregation, clipping of the peptide backbone, andoxidation of methionine residues. Physical instability encompasses manyphenomena, including, for example, aggregation and/or precipitation.

Chemical and physical stability can be promoted by removing water fromthe protein. Lyophilization (freeze-drying under controlled conditions)is commonly used for long-term storage of proteins. The lyophilizedprotein is substantially resistant to degradation, aggregation,oxidation, and other degenerative processes while in the freeze-driedstate. The lyophilized protein may be reconstituted with wateroptionally containing a bacteriostatic preservative (e.g., benzylalcohol) prior to administration.

Definitions

The term “carrier” includes a diluent, adjuvant, excipient, or vehiclewith which a composition is administered. Carriers can include sterileliquids, such as, for example, water and oils, including oils ofpetroleum, animal, vegetable or synthetic origin, such as, for example,peanut oil, soybean oil, mineral oil, sesame oil and the like.

The term “excipient” includes a non-therapeutic agent added to apharmaceutical composition to provide a desired consistency orstabilizing effect. Suitable pharmaceutical excipients include, forexample, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. [0045]The term “lyophilized” or “freeze-dried” includes astate of a substance that has been subjected to a drying procedure suchas lyophilization, where at least 90% of moisture has been removed.

VEGF Antagonists

A VEGF antagonist is a compound capable of blocking or inhibiting thebiological action of vascular endothelial growth factor (VEGF), andincludes fusion proteins capable of trapping VEGF. In a preferredembodiment, the VEGF antagonist is the fusion protein of SEQ ID NO:2 or4; more preferably, SEQ ID NO:4. In specific embodiments, the VEGFantagonist is expressed in a mammalian cell line such as a CHO cell andmay be modified post-translationally. In a specific embodiment, thefusion protein comprises amino acids 27-457 of SEQ ID NO:4 and isglycosylated at Asn residues 62, 94, 149, 222 and 308. Preferably, theVEGF antagonist is a dimer composed of two fusion proteins of SEQ IDNO:4.

The VEGF antagonist of the methods and formulations of the invention canbe prepared by any suitable method known in the art, or that comes to beknown. The VEGF antagonist is preferably substantially free of proteincontaminants at the time it is used to prepare the pharmaceuticallyacceptable formulation. By “substantially free of protein contaminants”is meant, preferably, that at least 90% of the weight of protein of theVEGF-specific fusion protein antagonist preparation used for making aformulation is VEGF fusion protein antagonist protein, more preferablyat least 95%, most preferably at least 99%. The fusion protein ispreferably substantially free of aggregates. “Substantially free ofaggregates” means that at least 90% of the weight of fusion protein isnot present in an aggregate at the time the fusion protein is used toprepare the pharmaceutically effective formulation. Unless statedotherwise, the phosphates employed are sodium phosphates and a desiredbuffering pH is achieved by mixing appropriate amounts of mono- anddibasic sodium phosphate.

Stable Liquid Ophthalmic Formulations

In one aspect, the invention provides a stable pharmaceuticallyacceptable formulation comprising a VEGF antagonist, wherein theformulation is a liquid formulation suitable for ophthalmic use.Preferably, the liquid formulation comprises a pharmaceuticallyeffective amount of the VEGF antagonist. The formulation can alsocomprise one or more pharmaceutically acceptable carriers, buffers,tonicity agents, stabilizers, and/or excipients. An example of apharmaceutically acceptable liquid formulation comprises a VEGFantagonist in a pharmaceutically effective amount, a buffer, an organicco-solvent such as polysorbate, a tonicity agent such as NaCl, andoptionally, a stabilizer such as sucrose or trehalose.

Stability is determined in a number of ways at specified time points,including determination of pH, visual inspection of color andappearance, determination of total protein content by methods known inthe art, e.g., UV spectroscopy, and purity is determined by, forexample, SDS-PAGE, size-exclusion HPLC, bioassay determination ofactivity, isoelectric focusing, and isoaspartate quantification. In oneexample of a bioassay useful for determining VEGF antagonist activity, aBAF/3 VEGFR1/EPOR cell line is used to determine VEGF165 binding by theVEGF antagonist of the invention.

Liquid formulations can be stored in an oxygen-deprived environment.Oxygen-deprived environments can be generated by storing theformulations under an inert gas such as, for example, nitrogen or argon.Liquid formulations are preferably stored at about 5° C.

Ophthalmic Lyophilized Formulations

In one aspect of the invention, an ophthalmically acceptable formulationcomprising a VEGF antagonist is provided, wherein the formulation is alyophilizable formulation. Lyophilizable formulations can bereconstituted into solutions, suspensions, emulsions, or any othersuitable form for administration or use. Lyophilizable formulations aretypically first prepared as liquids, then frozen and lyophilized. Thetotal liquid volume before lyophilization can be less, equal to, or morethan, the final reconstituted volume of the lyophilized formulation. Thelyophilization process is well known to those of ordinary skill in theart, and typically includes sublimation of water from a frozenformulation under controlled conditions.

Lyophilized formulations can be stored at a wide range of temperatures.Lyophilized formulations may be stored below 25° C., for example,refrigerated at 2-8° C., or at room temperature (e.g., approximately 25°C.). Preferably, lyophilized formulations are stored below about 25° C.,more preferably, at about 4-20° C.; below about 4° C.; below about −20°C.; about −40° C.; about −70° C., or about −80° C. Stability of thelyophilized formulation may be determined in a number of ways known tothe art, for example, by visual appearance of the cake and/or bymoisture content.

Lyophilized formulations are typically reconstituted for use by additionof an aqueous solution to dissolve the lyophilized formulation. A widevariety of aqueous solutions can be used to reconstitute a lyophilizedformulation. Preferably, lyophilized formulations are reconstitutedusing water. Lyophilized formulations are preferably reconstituted witha solution consisting essentially of water (e.g., USP WFI, or water forinjection) or bacteriostatic water (e.g., USP WFI with 0.9% benzylalcohol). However, solutions comprising buffers and/or excipients and/orone or more pharmaceutically acceptable carries can also be used.

Freeze-dried or lyophilized formulations are typically prepared fromliquids, that is, from solutions, suspensions, emulsions, and the like.Thus, the liquid that is to undergo freeze-drying or lyophilizationpreferably comprises all components desired in a final reconstitutedliquid formulation. As a result, when reconstituted, the freeze-dried orlyophilized formulation will render a desired liquid formulation uponreconstitution.

EXAMPLES

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly to the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

Example 1 Stability of 50 mg/ml VEGF Trap Liquid Formulation Stored at5° C. in 3 ml Glass Vials

An ophthalmic liquid formulation containing 50 mg/ml VEGF Trap (SEQ IDNO:4), 10 mM phosphate, 50 mM NaCl, 0.1% polysorbate 20, 5% sucrose, andpH 6.25, was stored at 5° C. in 3 ml glass vials and samples tested at3, 6, 9, 12, 18 and 24 months. Stability was determined by SE-HPLC. Theresults are shown in Table 1. Turbidity was measured at OD₄₀₅ nm; andpercent recovered protein and purity by size exclusion HPLC.

TABLE 1 Stability of 50 mg/ml VEGF Trap Protein (VGFT-SS065) % VEGF %VEGF Trap Visual Turbidity Trap Native Months Appearance (OD₄₀₅ nm) pHRecovered Configuration 0 Pass 0.00 6.2 100 98.8 3 Pass 0.00 6.2 10198.7 6 Pass 0.01 6.3 100 98.3 9 Pass 0.01 6.3 101 98.3 12 Pass 0.01 6.3104 98.4 18 Pass 0.01 6.3 96 98.1 24 Pass 0.01 6.3 105 98.1

Example 2 Stability of 50 mg/ml VEGF Trap Liquid Formulation Stored at5° C. in 3 ml Glass Vials

A liquid formulation containing 50 mg/ml VEGF Trap (SEQ ID NO:4), 10 mMphosphate, 50 mM NaCl, 3% polyethylene glycol 3350, 5% sucrose, and pH6.25, was stored at 5° C. in 3 nil glass vials and samples tested at 3,6, 9, 12, 18 and 24 months. Stability results are shown in Table 2.Turbidity, percent recovered protein and purity was determined asdescribed above.

TABLE 2 Stability of 50 mg/ml VEGF Trap Protein (VGFT-SS065) % VEGF %VEGF Trap Visual Trap Native Months Appearance Turbidity pH RecoveredConfiguration 0 Pass 0.00 6.2 100 98.9 3 Pass 0.00 6.1 104 98.5 6 Pass0.01 6.3 99 98.3 9 Pass 0.00 6.3 102 97.6 12 Pass 0.01 6.3 103 98.0 18Pass 0.00 6.3 113 97.7 24 Pass 0.00 6.2 106 97.6

Example 3 Stability of 40 mg/ml VEGF Trap Liquid Formulation Stored at5° C. in 3 ml Glass Vials

A liquid formulation containing 40 mg/ml VEGF Trap (SEQ ID NO:4), 10 mMphosphate, 40 mM NaCl, 0.03% polysorbate 20, 5% sucrose, and pH 6.3, wasstored at 5° C. in 3 ml glass vials and samples tested at 0.5, 1, 2, 3,and 4 months. Stability results are shown in Table 3. Turbidity, percentrecovered protein and purity was determined as described above.

TABLE 3 Stability of 40 mg/ml VEGF Trap Protein (VGFT-SS207) % VEGF %VEGF Trap Visual Trap Native Months Appearance Turbidity pH RecoveredConfiguration 0 Pass 0.00 6.3 100 99.5 0.5 Pass 0.00 6.3 99 99.4 1 Pass0.00 6.2 98 99.5 2 Pass 0.00 6.2 95 99.2 3 Pass 0.01 6.4 4 Pass 0.01 6.3

Example 4 Stability of 40 mg/ml VEGF Trap Liquid Formulation Stored at5° C. in Pre-filled Glass Syringe

A liquid formulation containing 40 mg/ml VEGF trap (SEQ ID NO:4), 10 mMphosphate, 40 mM NaCl, 0.03% polysorbate 20, 5% sucrose, and pH 6.3, wasstored at 5° C. in 1 ml prefilled luer glass syringe with 4023/50FluroTec coated plunger and samples tested at 0.5, 1, 2, 3, and 4months. Stability results are shown in Table 4. Turbidity, percentrecovered protein and purity was determined as described above.

TABLE 4 Stability of 40 mg/ml VEGF Trap Protein (VGFT-SS207) % VEGF %VEGF Trap Visual Trap Native Months Appearance Turbidity pH RecoveredConfiguration 0 Pass 0.00 6.3 100 99.4 0.5 Pass 0.00 6.3 100 99.3 1 Pass0.00 6.3 100 99.4 2 Pass 0.00 6.3 97 99.1 3 Pass 0.01 6.4 4 Pass 0.016.3

Example 5 Stability of 40 mg/ml VEGF Trap Liquid Formulation Stored at5° C. in 3 ml Glass Vials

A liquid formulation containing 40 mg/ml VEGF trap (SEQ ID NO:4), 10 mMphosphate, 135 mM NaCl, 0.03% polysorbate 20, and pH 6.3, was stored at5° C. in 3 ml glass vials and samples tested at 0.5, 1, 2, 3, and 4months. Stability results are shown in Table 5. Turbidity, percentrecovered protein and purity was determined as described above.

TABLE 5 Stability of 40 mg/ml VEGF Trap Protein (VGFT-SS203) % VEGF %VEGF Trap Visual Trap Native Months Appearance Turbidity pH RecoveredConfiguration 0 Pass 0.00 6.3 100 99.3 0.5 Pass 0.00 6.2 87 99.2 1 Pass0.00 6.2 88 99.1 2 Pass 0.00 6.3 103 99.2 3 Pass 0.00 6.3 88 99.0 4 Pass0.00 6.2 85 98.9 5 Pass 0.00 6.3 84 99.0

Example 6 Stability of 40 mg/ml VEGF Trap Liquid Formulation Stored at5° C. in 1 ml Pre-filled Glass Syringe

A liquid formulation containing 40 mg/ml VEGF trap (SEQ ID NO:4), 10 mMphosphate, 135 mM NaCl, 0.03% polysorbate 20, and pH 6.3, was stored at5° C. in 1 ml prefilled glass luer syringe with 4023/50 FluroTec coatedplunger and samples tested at 0.5, 1, 2, 3, 4, and 5 months. Stabilityresults are shown in Table 6. Turbidity, percent recovered protein andpurity was determined as described above.

TABLE 6 Stability of 40 mg/ml VEGF Trap Protein (VGFT-SS203) % VEGF %VEGF Trap Visual Trap Native Months Appearance Turbidity pH RecoveredConfiguration 0 Pass 0.00 6.3 100 99.2 0.5 Pass 0.01 6.3 101 99.2 1 Pass0.00 6.3 101 99.2 2 Pass 0.00 6.3 — — 3 Pass 0.01 6.3 102 99.1 4 Pass0.01 6.3 103 98.8 5 Pass 0.00 6.3  99 98.9

Example 7 Stability of Lyophilized 20 mg/ml VEGF Trap Formulation Storedat 5° C. in 3 ml Glass Vials and Reconstituted to 40 mg/ml

0.8 ml of a liquid formulation containing 20 mg/ml VEGF trap (SEQ IDNO:4), 5 mM phosphate, 20 mM NaCl, 0.015% polysorbate 20, 2.5% sucrose,and pH 6.3, were lyophilized in 3 ml glass vials. Samples were stored at5° C. and tested at 1, and 2 months. VEGF trap was reconstituted to afinal concentration of 40 mg/ml VEGF Trap (final volume of 0.4 ml).Stability results are shown in Table 7 (t=time in months; *=visualappearance; **=reconstitution time). Turbidity, percent recoveredprotein and purity was determined as described above.

TABLE 7 Stability of Lyophilized 20 mg/ml VEGF Trap Protein (VGFT-SS216)Recon. Vis. App.* Vis. Time** Reconst'd % VEGF Trap % VEGF Trap t App.*(min) Liquid Turbidity pH Recovered Native Config. 0 Pass 0.6 Pass 0.006.3 100 99.5 1 Pass 0.6 Pass 0.01 6.3 106 99.4 2 Pass 0.4 Pass 0.01 6.2103 99.3

Example 8 Stability of Lyophilized 20 mg/ml VEGF Trap Formulation Storedat 5° C. in 3 ml Glass Vials

0.8 ml of a liquid formulation containing 20 mg/ml VEGF trap (SEQ IDNO:4), 5 mM phosphate, 67.5 mM NaCl, 0.015% polysorbate 20, and pH 6.3,were lyophilized in 3 ml glass vials. Samples were stored at 5° C. andtested at 1, 2, and 3 months. VEGF trap was reconstituted to a finalconcentration of 40 mg/ml VEGF trap (final volume of 0.4 ml). Stabilityresults are shown in Table 8 (t=time in months; *=visual appearance;**=reconstitution time).

TABLE 8 Stability of Lyophilized 20 mg/ml VEGF Trap Protein (VGFT-SS216)Recon. Vis. App. % VEGF Time** Reconst'd Trap % VEGF Trap t Vis. App.*(min) Liquid Turbidity pH Recovered Native Config. 0 Pass 0.7 Pass 0.006.3 100 99.0 1 Pass 0.7 Pass 0.01 6.2 105 98.9 2 Pass 0.4 Pass 0.01 6.2103 98.9

We claim:
 1. A method of producing a lyophilized formulation of a VEGFantagonist, comprising a step of subjecting to lyophilization apre-lyophilized formulation, which comprises: (a) 5-50 mg/ml of avascular endothelial growth factor (VEGF) antagonist, comprising theamino acids 27-457 of SEQ ID NO:4; (b) 5-25 mM of sodium phosphatebuffer, pH about 5.8-7.0; (c) 0.01-0.15% of an organic co-solvent,selected from the group consisting of polysorbate, polythylene glycol(PEG) propylene glycol, and a combination thereof; and, (d) 1-10% of astabilizing agent selected from the group consisting of sucrose,sorbitol, glycerol, trehalose, and mannitol, to generate a lyophilizedformulation.
 2. The method of claim 1, wherein the prelyophilizationformulation comprises about 20 mg/ml of the VEGF antagonist, about 10 mMsodium phosphate buffer, about 0.03% polysorbate, about 0.1% PEG, andabout 2.5% sucrose, pH about 6.2-6.3.
 3. The method of claim 1, whereinthe prelyophilization formulation comprises about 20 mg/ml of the VEGFantagonist, about 5 mM sodium phosphate buffer, about 0.015%polysorbate, about 2.5% sucrose, and further comprising sodium chlorideat about 20 mM, pH about 6.2-6.3.
 4. The method of claim 1, wherein theprelyophilization formulation comprises about 20 mg/ml of the VEGFantagonist, about 5 mM sodium phosphate buffer, about 0.015%polysorbate, and further comprising sodium chloride at about 67.5 mM, pHabout 6.2-6.3.
 5. A lyophilized formulation made according to the methodof claim
 1. 6. The lyophilized formulation of claim 5, wherein theprelyophilization formulation comprises about 20 mg/ml of the VEGFantagonist, about 10 mM sodium phosphate buffer, about 0.03%polysorbate, about 0.1% PEG, and about 2.5% sucrose, pH about 6.2-6.3.7. The lyophilized formulation of claim 5, wherein the prelyophilizationformulation comprises about 20 mg/ml of the VEGF antagonist, about 5 mMsodium phosphate buffer, about 0.015% polysorbate, about 2.5% sucrose,and further comprising sodium chloride at about 20 mM, pH about 6.2-6.3.8. The lyophilized formulation of claim 5, wherein the prelyophilizationformulation comprises about 20 mg/ml of the VEGF antagonist, about 5 mMsodium phosphate buffer, about 0.015% polysorbate, and furthercomprising sodium chloride at about 67.5 mM, pH about 6.2-6.3.
 9. Amethod of producing a lyophilized formulation of a VEGF antagonist,comprising the step of subjecting to lyophilization a prelyophilizationformulation, which comprises a) 5-50 mg/ml of a vascular endothelialgrowth factor (VEGF) antagonist, comprising amino acids 27-457 of SEQ IDNO:4; (b) 5-25 mM of a phosphate buffer, pH about 5.8-7.0; (c)0.01-0.15% of an organic co-solvent, selected from the group consistingof polysorbate, polyethylene glycol (PEG), propylene glycol, and acombination thereof; and (d) 20-150 mM of a tonicity agent, to generatea lyophilized formulation.
 10. The method of claim 9, wherein thetonicity agent is sodium chloride at a concentration selected from thegroup consisting of 20 mM and 67.5 mM.
 11. The method of claim 9,wherein the organic co-solvent is polysorbate at 0.015%.
 12. The methodof claim 9, wherein the VEGF antagonist is at a concentration selectedfrom the group consisting of 5 mg/ml, 10 mg/ml, 20 mg/ml, and 40 mg/ml.13. The method of claim 9, wherein the sodium phosphate buffer is at 5mM.
 14. A lyophilized formulation made according to the method of claim9.
 15. The lyophilized formulation of claim 14, wherein the tonicityagent is sodium chloride at a concentration selected from the groupconsisting of 20 mM and 67.5 mM.
 16. The lyophilized formulation ofclaim 14, wherein the organic co-solvent is polysorbate at 0.015%. 17.The lyophilized formulation of claim 14, wherein the VEGF antagonist isat a concentration selected from the group consisting of 5 mg/ml, 10mg/ml, 20 mg/ml, and 40 mg/ml.
 18. The lyophilized formulation of claim14, wherein the sodium phosphate buffer is at 5 mM.